Muscular dysgenesis is a murine disorder, which results from a single lethal, recessive, autosomal mutation (mdg), characterized by progressive degeneration of embryonic skeletal muscle and lack of muscle contraction. The mdg gene has been identified as the altered gene for the alpha 1 subunit, of the dihydropyridine (DHP) receptor/slow calcium channel, found in the t-tubule membrane of normal muscle. The lack of this subunit (and thus the slow calcium current) results in an arrest of myogenesis, including incomplete fibrillogenesis and normal triad formation, triadogenesis. Dysgenic muscle is also highly fusogenic; nonmyogenic cells (embryonic spinal cord cells and fibroblasts) fuse with mutant myotubes and the muscle is rescued with respect to contraction and fine-structural maturation. The specific aims of this project relate directly to the mechanism of fusion of dysgenic (and normal) cells and the resultant rescue domains of dysgenic muscle: 1. Clarification of the cell types involved in spinal cord cell fusion with dysgenic muscle, and determination of the dominant or semi- dominant nature of the enhanced fusibility characteristic of dysgenic muscle. Heterospecific (rat) fibroblasts or Schwann cells will be added to developing myotubes (mdg/mdg, +/mdg and +/+) and the incidence of fusion monitored cytologically by using the nuclear Hoechst stain. Biochemical quantification of the fusion will be determined by the production of chloramphenicol acetyltransferase (CAT) activity following addition of similar cell types from transgenic mice carrying a CAT reporter gene driven by a skeletal muscle specific promoter. 2. Study of the fusion process (myoblast/myoblast; myoblast/myotube) in normal and dysgenic muscle. An analysis of fusion will be studied by time lapse video microscopy to describe a recently noted bubbling phenomenon characteristic of the fusion process. Immunocytological techniques at the light and SEM levels will be used to identify the molecular topology of fusing membrane areas. 3. The description of non-myogenic (eg.fibroblast) cell fusion with dysgenic myotubes, utilizing techniques described above. 4. A study of the extent of rescued dysgenic myotube domains. The triadic and sarcomeric structures of the rescued areas will be revealed at the light microscopic level by conventional epifluorescent optics as well as by confocal laser optics, and at the TEM and SEM level with immunogold techniques. These studies should increase the understanding of the mechanisms of normal myogenic cell fusion, as well as enhanced fusion in dysgenic muscle. The studies of nuclear domains, relevant to an understanding of myofiber specialization (synapse formation) in normal muscle, may have important implications for fibroblastic cell rescue in human muscle diseases such as the muscular dystrophies.